Traumatic brain injury (TBI) is a major cause of morbidity among the Veteran population. Mild TBI (mTBI) has been associated with substance abuse as a comorbid condition, hampering rehabilitation and treatment efforts of either condition. Of particular interest is the rising trend of opiate abuse among Veterans. Existing evidence indicates overlapping molecular and neural pathways for TBI and opiate abuse; for example, inflammatory mediators and increased neurodegeneration and microglial activation are observed in both opiate abuse and mTBI. To the best of our knowledge, there are no reported preclinical studies that have systematically examined the crosstalk between mTBI and subsequent opiate exposure in terms of neurobehavioral, neuropathological and neurobiochemical consequences. This study proposes to use a validated rodent model of repeated closed head injury (rCHI) paired with chronic exposure to fentanyl drug to closely simulate real-world conditions of mTBI and opiate usage. Fentanyl is a highly effective but addictive analgesic commonly used for treating chronic pain. The central hypothesis of this proposal is that sustained opiate exposure post-mTBI will induce characteristic neurofunctional and neuropathological changes that differ from those seen with mTBI or opiate exposure alone, and that these changes can be identified and evaluated via neurobehavioral, biochemical and neuroproteomic approaches. This proposal is a new direction for the PI?s laboratory and is potentially paradigm-shifting. Aim 1 will determine the effects of 1 month fentanyl opiate exposure followed by 1 month withdrawal after r-CHI on cognition, pain sensation, motor activity and spontaneous opiate withdrawal symptoms. Male mice (C57BL6) will be used and subjected to rCHI or sham procedure (non-injury).There will be four experimental groups: (a) Sham+saline, (b) rCHI+saline, (c) sham+fentanyl, (d) rCHI-followed by Fentanyl exposure. Functional assessment tests include: TBI-related cognition (spatial memory performance; Morris water maze) and anxiety- behavior (elevated plus maze), or opiate-use/withdrawal tests; locomotive activity-related pain sensitivity tests (warm-water tail withdrawal and Orofacial formalin test) (during drug exposure and withdrawal phases), and drug withdrawal-dependence symptoms (during the fentanyl withdrawal phase). The results will be compared with the observations from rCHI or fentanyl exposure alone. Aim 2 will assess key neuropathological, neural cellular markers and opiate receptor levels in three brain regions (cortex, hippocampus and ventral tegmental area) implicated in TBI and/or opioid receptor-mediated reward circuits. Brain samples will be analyzed at 2 endpoints - at the end of fentanyl administration (month 1), and after 1 month of withdrawal (month 2), in animals with or without rCHI. Neuronal, axonal, astroglial, microglial, synaptic and white matter markers, along with and the three subtypes of opioid receptors will be assessed in separate sets of animals by immunoblotting/ELISA essays and by immunohistochemical-histopathological analyses. Aim 3 will identify long-term mTBI consequences and opiate abuse comorbidity-related pathological pathways/key drivers as putative neurotherapeutic targets using neuroproteomic and System Biology analysis. Collectively, this new line of investigation aims to provide insights into the neurobiological and neuropathological interplay between mTBI and post injury opioid abuse. The results may guide possible new therapeutic strategies to treat TBI and comorbid opioid dependence in Veterans.